Induced DNA Lesions Research Articles

Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF) at 5mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50μM or 1mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5mM NaF together with radiation (1Gy) induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

Physical exercise and redox balance in type 2 diabetics: effects of moderate training on biomarkers of oxidative stress and DNA damage evaluated through comet assay.

Objective. Hyperglycemia leads to increased production of reactive oxygen species (ROS) in type 2 diabetes, which reduces cellular antioxidant defenses and induces DNA lesions. The aim of this study was to investigate the effects on redox homeostasis and DNA oxidative damage of exercise training in patients with type 2 diabetes compared with nondiabetic individuals. Methods and Results. 12 sedentary type 2 diabetic males (62.1 ± 4.3 yrs) and 12 sedentary healthy males (61.7 ± 3.9 yrs) were exposed to 4-month moderate training, 3 times per week, to evaluate the effect on plasma biomarkers of oxidative stress malondialdehyde and antioxidant status (GSSG, GSH/GSSG, and ascorbic acid) as well as basal and H2O2-induced DNA damage trough alkaline comet assay in peripheral blood lymphocytes. After training, glutathione and ascorbic acid levels increased in both groups, but only in diabetics the malondialdehyde as well as the DNA damage decreased. Conclusion. Our study demonstrates for the first time that moderate exercise training is not only effective in improving the redox homeostasis, through an increase of the endogenous antioxidant defences in healthy as well as in diabetic patients, but also, specifically in diabetic patients, effective in lowering the susceptibility to oxidative DNA damage and the lipid peroxidation levels.

Chromium (VI)-induced transformation is enhanced by Zn deficiency in BALB/c 3T3 cells.

Hexavalent chromium [Cr(VI)] is a carcinogenic heavy metal that is reduced to intermediate oxidation states, such as Cr(V) and Cr(IV), in the process of forming stable Cr(III) forms; it is these intermediate forms that are thought to be responsible for much of the DNA damage and mutations that are induced by Cr(VI). Metallothionein (MT), a heavy metal-binding protein, is induced by zinc and other heavy metals and protects cells from the toxic effects of these metals by sequestering them. MT cannot bind Cr, but by scavenging reactive oxygen species through its cysteine residues, it may act as a protective factor against Cr(VI)-induced DNA lesions by reducing Cr(VI) directly to Cr(III), thereby avoiding the creation of the toxic intermediates. Here, we showed that Zn deficiency decreased MT expression in BALB/3T3 clone A31-1-1 cells and caused them to become highly susceptible to Cr(VI)-induced transformation. To obtain Zn-deficient cultures, cells were cultured in medium supplemented with 10% Chelex(®)-100 chelating resin-treated FBS. The increase in susceptibility to transformation was abolished by culturing the cells with supplemental Zn (50 µM). Previously, we reported that Cr(VI) inhibits MT transcription by preventing the zinc-dependent formation of a complex of metal response element-binding transcription factor-1 (MTF-1) and the co-activator p300. Our results suggest that the carcinogenicity of Cr(VI) is enhanced by MTF-1 dysfunction.

HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs.

BackgroundThe repair of spontaneous and induced DNA lesions is a multistep process. Depending on the type of injury, damaged DNA is recognized by many proteins specifically involved in distinct DNA repair pathways.ResultsWe analyzed the DNA-damage response after ultraviolet A (UVA) and γ irradiation of mouse embryonic fibroblasts and focused on upstream binding factor 1 (UBF1), a key protein in the regulation of ribosomal gene transcription. We found that UBF1, but not nucleolar proteins RPA194, TCOF, or fibrillarin, was recruited to UVA-irradiated chromatin concurrently with an increase in heterochromatin protein 1β (HP1β) level. Moreover, Förster Resonance Energy Transfer (FRET) confirmed interaction between UBF1 and HP1β that was dependent on a functional chromo shadow domain of HP1β. Thus, overexpression of HP1β with a deleted chromo shadow domain had a dominant-negative effect on UBF1 recruitment to UVA-damaged chromatin. Transcription factor UBF1 also interacted directly with DNA inside the nucleolus but no interaction of UBF1 and DNA was confirmed outside the nucleolus, where UBF1 recruitment to DNA lesions appeared simultaneously with cyclobutane pyrimidine dimers; this occurrence was cell-cycle-independent.ConclusionsWe propose that the simultaneous presence and interaction of UBF1 and HP1β at DNA lesions is activated by the presence of cyclobutane pyrimidine dimers and mediated by the chromo shadow domain of HP1β. This might have functional significance for nucleotide excision repair.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-8935-7-39) contains supplementary material, which is available to authorized users.

Genotoxic effects of two-generational selenium deficiency in mouse somatic and testicular cells.

Many studies have investigated genotoxic effects of high Se diets but very few have addressed the genotoxicity of Se deprivation and its consequences in germ cells and none in somatic cells. To address these data gaps, C57BL/6 male mice were subjected to Se deprivation starting in the parental generation, i.e. before conception. Mice were given a diet of either low (0.01mg Se/kg diet) or normal (0.23mg Se/kg diet) Se content. Ogg1-deficient (Ogg1 (-/-) ) mice were used as a sensitive model towards oxidative stress due to their reduced capacity to repair oxidised purines. Ogg1 (-/-) mice also mimic the repair characteristics of human post-meiotic male germ cells which have a reduced ability to repair such lesions. The genotoxicity of Se deficiency was addressed by measuring DNA lesions with the alkaline single cell gel electrophoresis (+ Fpg to detect oxidised DNA lesions) in somatic cells (nucleated blood cells and lung cells) and male germ cells (testicular cells). Total Se concentration in liver and GPx activity in plasma and testicular cells were measured. Gene mutation was evaluated by an erythrocyte-based Pig-a assay. We found that Se deprivation of F1 from their conception and until early adulthood led to the induction of DNA lesions in testicular and lung cells expressed as significantly increased levels of DNA lesions, irrespective of the mouse genotype. In blood cells, Se levels did not appear to affect DNA lesions or mutant cell frequencies. The results suggest that the testis was the most sensitive tissue. Thus, genotoxicity induced by the low Se diet in the spermatozoal genome has potential implications for the offspring.

Dynamics of polycomb proteins-mediated histone modifications during UV irradiation-induced DNA damage

Polycomb group (PcG) complexes are known to be chromatin modifiers and transcriptional repressors. In this work, we reported that the histone-modifying PcG complexes are able to participate in the repair process of ultraviolet (UV)-induced DNA lesions in the silkworm, Bombyx mori. The silkworm cells with depletion of PcG genes showed hypersensitive to UV–C irradiation and increased inhibition of cell proliferation. Interestingly, an SQ site in the silkworm-human chimeric H2A protein synthesized here was phosphorylated rapidly upon UV–C exposure, which could be used as a marker for monitoring the response to DNA damage in silkworm cells. Under these UV–C irradiated conditions, we found that PRC1-mediated ubiquitylation of H2AX, but not of H2AZ, were decreased and this deubiquitylation was independent of its phosphorylation event. In contrast, UV–C irradiation induced the increase of trimethylation of lysine 27 on histone H3 (H3K27me3), a mark of transcriptionally silent chromatin catalyzed by another PcG subcomplex, PRC2. Collectively, we provided the first evidence on chromatin remodeling in response to UV–C lesion in silkworm and revealed another layer role for PcG complexes-mediated histone modifications in contributing to creating an open chromatin structure for the efficient repair of DNA damages.

Abstract 5089: Survivin safeguards chromosome numbers and protects from aneuploidy

Abstract Survivin, belonging to the family of Inhibitor of Apoptosis proteins (IAP), is overexpressed in a broad variety of different tumor entities. Besides its role and function in apoptosis, Survivin forms in mitosis with its partners Borealin, inner centromere protein (INCENP) and the enzymatically active member Aurora B kinase the chromosomal passenger complex (CPC). The main function of this complex is sensing and correcting non-bipolar microtubule-kinetochore interaction, regulating chromosome segregation and cytokinesis. Some evidence points to a role of Survivin in connecting mitosis with control of cell cycle arrest. In this study, we assessed the role of Survivin in ensuring chromosomal stability and DNA integrity in HCT116, MCF-7, U87-MG and corresponding isogenic p53-deficient cells. Knock down of Survivin, using retroviral short-hairpin RNAs, caused in all tested cell lines a heavily impaired cytokinesis resulting in polyploidy and poly-merotelic kinetochore spindle assemblies, irrespective of the p53 status. Surprisingly, cells with wild type p53 showed after Survivin RNAi an activation of p53 and accumulation of p21waf/cip and Cyclin D1, indicating G1 arrest. A detailed Flow Cytometry analysis revealed a correlation between the increased p21waf/cip expression and increasing DNA content in Survivin-depleted wild type cells compared to p53-deficient cells. Conversely, BrdU incorporation in wild type cells decreased with DNA content compared to p53-deficient cells with knock down of Survivin. Nonetheless, polyploid wild type cells even with a DNA content larger 4n were still able to incorporate BrdU, indicating transient G1 arrest after Survivin knock down. Furthermore, Survivin RNAi led in all tested cell lines to an induction of DNA lesions and caused a DNA damage response. SKY-analysis revealed numerical aberrations and a significant higher frequency of structural chromosomal aberrations indicative for DNA double strand break repair, only in Survivin-depleted cells. In conclusion, our results highlight the important role of Survivin as a chromosomal passenger protein which in concert with its molecular partners Aurora B, INCENP, and Borealin, controls chromosomal segregation by regulating bipolar spindle attachment and cytokinesis and protects cells from polyploidy and chromosomal instability. Citation Format: Ralf Wiedemuth, Barbara Klink, Evelin Schroeck, Gabriele Schackert, Achim Temme. Survivin safeguards chromosome numbers and protects from aneuploidy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5089. doi:10.1158/1538-7445.AM2014-5089

DNA end resection is needed for the repair of complex lesions in G1-phase human cells

ABSTRACTRepair of DNA double strand breaks (DSBs) is influenced by the chemical complexity of the lesion. Clustered lesions (complex DSBs) are generally considered more difficult to repair and responsible for early and late cellular effects after exposure to genotoxic agents. Resection is commonly used by the cells as part of the homologous recombination (HR) pathway in S- and G2-phase. In contrast, DNA resection in G1-phase may lead to an error-prone microhomology-mediated end joining. We induced DNA lesions with a wide range of complexity by irradiation of mammalian cells with X-rays or accelerated ions of different velocity and mass. We found replication protein A (RPA) foci indicating DSB resection both in S/G2- and G1-cells, and the fraction of resection-positive cells correlates with the severity of lesion complexity throughout the cell cycle. Besides RPA, Ataxia telangiectasia and Rad3-related (ATR) was recruited to complex DSBs both in S/G2- and G1-cells. Resection of complex DSBs is driven by meiotic recombination 11 homolog A (MRE11), CTBP-interacting protein (CtIP), and exonuclease 1 (EXO1) but seems not controlled by the Ku heterodimer or by phosphorylation of H2AX. Reduced resection capacity by CtIP depletion increased cell killing and the fraction of unrepaired DSBs after exposure to densely ionizing heavy ions, but not to X-rays. We conclude that in mammalian cells resection is essential for repair of complex DSBs in all phases of the cell-cycle and targeting this process sensitizes mammalian cells to cytotoxic agents inducing clustered breaks, such as in heavy-ion cancer therapy.

Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting

The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

Assessment of antioxidative, chelating, and DNA-protective effects of selected essential oil components (eugenol, carvacrol, thymol, borneol, eucalyptol) of plants and intact Rosmarinus officinalis oil.

Selected components of plant essential oils and intact Rosmarinus officinalis oil (RO) were investigated for their antioxidant, iron-chelating, and DNA-protective effects. Antioxidant activities were assessed using four different techniques. DNA-protective effects on human hepatoma HepG2 cells and plasmid DNA were evaluated with the help of the comet assay and the DNA topology test, respectively. It was observed that whereas eugenol, carvacrol, and thymol showed high antioxidative effectiveness in all assays used, RO manifested only antiradical effect and borneol and eucalyptol did not express antioxidant activity at all. DNA-protective ability against hydrogen peroxide (H2O2)-induced DNA lesions was manifested by two antioxidants (carvacrol and thymol) and two compounds that do not show antioxidant effects (RO and borneol). Borneol was able to preserve not only DNA of HepG2 cells but also plasmid DNA against Fe(2+)-induced damage. This paper evaluates the results in the light of experiences of other scientists.

Molecular nature of chemically and physically induced mutants in plants: a review

More than 3200 new mutant varieties have been bred and used by millions of farmers, which has significantly contributed to world food security. A lot more mutants have also served as tools for gene discovery and functional analysis. Recent genomic approaches including TILLING (Targeting Induced Local Lesions In Genome) have enabled screening of mutations at the molecular level. This review describes the molecular nature of chemically and physically induced mutations and their repercussions. Analyses of TILLING reports indicate that chemically induced mutations are mostly nucleotide substitutions, but that mutation frequencies fluctuate among plant species ranging from one DNA lesion per 300 kbp inArabidopsisto one DNA lesion per 30 kbp in bread wheat, which reciprocate with an increase in ploidy levels. The majority (>95%) of chemically induced DNA lesions are silent or missense mutations. Mutations induced by physical mutagens seem to be more diverse, including single-nucleotide substitutions, insertions, inversions and translocations, although short deletions ( < 15 bp) are relatively more predominant. The proportion of complex mutations (translocation, inversions, etc.) may increase with an increase in the linear energy transfer of radiations. In addition, the implications of these findings for the roles of induced mutants in breeding and gene function analysis are briefly discussed.

Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities.

Loss of Werner syndrome protein function causes Werner syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN's DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor HU. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency.

Infrared laser effects at fluences used for treatment of dentin hypersensitivity on DNA repair in Escherichia coli and plasmids

Abstract Low-intensity infrared lasers are proposed in clinical protocols based on biostimulative effects, yet dosimetry is inaccurate and their effects on DNA at therapeutic doses are controversial. The aim of this work was to evaluate the effects of low-intensity infrared laser on survival and induction of filamentation of Escherichia coli cells, and induction of DNA lesions in bacterial plasmids. E. coli cultures were exposed to laser (808 nm, 100 mW, 40 and 60 J/cm2) to study bacterial survival and filamentation. Also, bacterial plasmids were exposed to laser to study DNA lesions by electrophoretic profile and action of DNA repair enzymes. Data indicate low-intensity infrared laser has no effect on survival of E. coli wild type and exonuclease III, but decreases the survival of formamidopyrimidine DNA glycosylase/MutM protein and endonuclease III deficient cells in stationary growth phase, induces bacterial filamentation, does not alter the electrophoretic profile of plasmids in agarose gels and does not alter the electrophoretic profile of plasmids incubated with endonuclease III, formamidopyrimidine DNA glycosylase/MutM protein and exonuclease III. Our findings show that low-intensity laser exposure causes DNA lesions at sub-lethal level and induces cellular mechanisms involved in repair of oxidative lesions in DNA. Studies about laser dosimetry and safety strategies are necessary for professionals and patients exposed to low-intensity lasers at therapeutic doses.

Site-directed Mutagenesis (Y52E) of POLH Affects Its Ability to Bypass Ultraviolet-induced DNA Lesions in HaCaT Cells.

DNA polymerase eta (Pol η) is one of several Y family trans-lesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased both with empty vector control (EVC), wild-type POLH, and Y52E-POLH over 48 hours post UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV induced DNA lesions in HaCaT cells.

Recruitment of HP1β to UVA‐induced DNA lesions is independent of radiation‐induced changes in A‐type lamins

The optimal repair of DNA lesions is fundamental for physiological processes. We asked whether the recruitment of HP1β, 53BP1 and BMI1 proteins to ultraviolet (UVA)-induced DNA lesions requires functional A-type lamins. We found that UVA irradiation of nuclear lamina abolished the fluorescence of mCherry-tagged A-type lamins and destroyed the nuclear lamina as also observed by electron microscopy studies. Similarly, an absence of endogenous A- and B-type lamins was found in irradiated regions by UVA. However, irradiation did not affect the recruitment of HP1β, 53BP1 and BMI1 to DNA lesions. The UVA-induced shrinkage of the nuclear lamina, which anchors chromatin, explains why UVA-micro-irradiated chromatin is relaxed. Conversely, additional experiments with γ-irradiation showed that the nuclear lamina remained intact and the genome-wide level of HP1β was stable. Fluorescence intensity of HP1β and BMI1 in UVA-induced DNA lesions and level of HP1β after γ-irradiation were unaffected by deficiency in A-type lamins, whereas those parameters of 53BP1 were changed. We conclude that only the 53BP1 status in DNA lesions, induced by UVA or γ-rays, is affected by A-type lamin deficiency, which was not observed for heterochromatin-related proteins HP1β and BMI1.

Characterization and differential expression of CPD and 6–4 DNA photolyases in Xiphophorus species and interspecies hybrids

Among the many Xiphophorus interspecies hybrid tumor models are those that exhibit ultraviolet light (UVB) induced melanoma. In previous studies, assessment of UVB induced DNA damage and nucleotide excision DNA repair has been performed in parental lines and interspecies hybrids. Species and hybrid specific differences in the levels of DNA damage induced and the dark repair rates for cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine pyrimidine photoproducts (6–4PPs) have been reported. However, UVB induced DNA lesions in Xiphophorus fishes are thought to primarily be repaired via light dependent CPD and 6–4PP specific photolyases. Photolyases are of evolutionary interest since they are ancient and presumably function solely to ameliorate the deleterious effects of UVB exposure. Herein, we report results from detailed studies of CPD and 6-4PP photolyase gene expression within several Xiphophorus tissues. We determined photolyase gene expression patterns before and after exposure to fluorescent light in X. maculatus, X. couchianus, and for F1 interspecies hybrids produced from crossing these two parental lines (X. maculatus Jp 163 B×X. couchianus). We present novel results showing these two photolyase genes exhibit species, tissue, and hybrid-specific differences in basal and light induced gene expression.

An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

Biological risk assessment studies of chemical substances that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication, but also with transcription. There is no simple method for the detection of the DNA lesion-induced inhibition of transcription. Here, we report an assay for estimating the toxicity of chemical substances by visualizing transcription in mammalian cells using nucleotide analog 5-ethynyluridine (EU) and its click chemistry reaction. Ultraviolet light and representative chemical substances (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) of DNA- damaging agents show toxicity, as indicated by RNA synthesis inhibition in response to DNA damage in HeLa cells. Using titanium dioxide, we observed RNA synthesis inhibition in response to the rutile form, but not the anatase form, indicating that rutile titanium dioxide is a toxic substance. Because this method is based on the transcriptional response to DNA lesions, we can use terminally differentiated neuron-like PC12 cells, the differentiation of which can be induced by nerve growth factors, for evaluating chemical substances. Ultraviolet light and some chemicals (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) inhibited RNA synthesis in non-differentiated PC12 cells. Conversely, camptothecin and cisplatin did not inhibit RNA synthesis in differentiated PC12 cells, but 4-nitroquinoline-1-oxide, mitomycin C, and etoposide did. And using titanium dioxide, we did not observed any RNA synthesis inhibition. These data suggest that this method might be used to estimate the potential risk of chemical substances in differentiated mammalian cells, which are the most common cell type found in the human body.

Bak and Mcl-1 are essential for Temozolomide induced cell death in human glioma.

Temozolomide (TMZ) is an alkylating agent used for the treatment of glioblastoma multiforme (GBM), the main form of human brain tumours in adults. It has been reported that TMZ induced DNA lesions that subsequently trigger cell death but the actual mechanisms involved in the process are still unclear. We investigated the implication of major proteins of the Bcl-2 family in TMZ-induced cell death in GBM cell lines at concentrations closed to that reached in the brain during the treatments. We did not observe modulation of autophagy at these concentrations but we found an induction of apoptosis. Using RNA interference, we showed that TMZ induced apoptosis is dependent on the pro-apoptotic protein Bak but independent of the pro-apoptotic protein Bax. Apoptosis was not enhanced by ABT-737, an inhibitor of Bcl-2/Bcl-Xl/Bcl-W but not Mcl-1. The knock-down of Mcl-1 expression increased TMZ induced apoptosis. Our results identify a Mcl-1/Bak axis for TMZ induced apoptosis in GBM and thus unravel a target to overcome therapeutic resistance toward TMZ.

Evaluation of DNA Damage Induced by Therapeutic Low-Level Red Laser

Biological effects of monochromatic lights on cells have aroused interest regarding an active and non-innocuous effect on human skin. The aim of this work was to evaluate DNA damage induced by low-level red laser at doses and frequencies used in therapeutic protocols. For this purpose, E. coli cultures and bacterial plasmids were used to assess bacterial survival, filamentation, DNA lesions and in vitro DNA repair induced by low-level red laser exposure at low doses in continuous wave and pulsed emission mode. Data indicate that low-level red laser does not affect the survival of E. coli cultures, topological forms of DNA, and does not induce DNA lesions targeted by endonuclease IV, formamidopyrimidine DNA glycosylase and endonuclease III, but rather that it induces bacterial filamentation in wild type and DNA repair-deficient E. coli cultures and DNA lesions targeted by exonuclease III. Monochromatic red light could activate survival and/or adaptive mechanisms against harmful radiations.

DNA repair diseases: What do they tell us about cancer and aging?

The discovery of DNA repair defects in human syndromes, initially in xeroderma pigmentosum (XP) but later in many others, led to striking observations on the association of molecular defects and patients' clinical phenotypes. For example, patients with syndromes resulting from defective nucleotide excision repair (NER) or translesion synthesis (TLS) present high levels of skin cancer in areas exposed to sunlight. However, some defects in NER also lead to more severe symptoms, such as developmental and neurological impairment and signs of premature aging. Skin cancer in XP patients is clearly associated with increased mutagenesis and genomic instability, reflecting the defective repair of DNA lesions. By analogy, more severe symptoms observed in NER-defective patients have also been associated with defective repair, likely involving cell death after transcription blockage of damaged templates. Endogenously induced DNA lesions, particularly through oxidative stress, have been identified as responsible for these severe pathologies. However, this association is not that clear and alternative explanations have been proposed. Despite high levels of exposure to intense sunlight, patients from tropical countries receive little attention or care, which likely also reflects the lack of understanding of how DNA damage causes cancer and premature aging.